Download Analysis and Characterization of Nucleic Acids and Proteins

Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Analysis and Characterization of Nucleic Acids and Proteins
Chapter 6
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Objectives
 Describe how restriction enzyme sites are mapped on DNA.
 Construct a restriction enzyme map of a DNA plasmid or fragment.
 Diagram the Southern blot procedure.  Define hybridization, stringency, and melting
temperature.
 Calculate the melting temperature of a given sequence of dsDNA.
 Describe comparative genomic hybridization (CGH).
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Restriction Enzyme Mapping
 Clinical and forensic analyses require characterization of specific genes or genomic regions at the molecular level. Because of their sequence‐specific activity, restriction endonucleases provide a convenient tool for molecular characterization of DNA
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Restriction Endonucleases
Enzyme
Isolated From
Recognition
Sequence
Eco RI
E. coli, strain R,
1st enzyme
Gν AATTC
Eco RV
E. coli, strain R,
5th enzyme
Gv ATATC
Hind III
H. influenzae, strain d,
3rd enzyme
Av AGCTT
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Restriction Enzymes
 Type I

methylation/cleavage (3 subunits)
>1000 bp from binding site
e.g., Eco AI
GAGNNNNNNNGTCA
 Type II

cleavage at specific recognition sites
 Type III

methylation/cleavage (2 subunits) 24–26 bp from binding site
e.g.,
Hinf III CGAAT
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Restriction Enzymes, Type II
5′
G AATTC
CTTAA G
5′
CCC GGG
GGG CCC
5′
Eco R1
5′ overhang
5′
Sma1
blunt
5′
CTGCA G
G ACGTC
5′
PstI
3′ overhang
Restriction enzymes cut DNA at specific sites in one of three ways.
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Sticky ends must match (be complementary) for optimal religation.
Blunt ends can be religated with less efficiency than sticky ends.
Sticky ends can be converted to blunt ends with nuclease or
polymerase.
Blunt ends can be converted to sticky ends by ligating to synthetic
adaptors.
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Restriction Enzyme Mapping
 Digest DNA with a restriction enzyme.
 Resolve the fragments by gel electrophoresis.
 The number of bands indicates the number of restriction sites.
 The size of the bands indicates the distance between restriction sites.
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
Restriction mapping of a linear DNA fragment (top green bar). The fragment is first cut with the enzyme PstI. Four fragments result, as deter‐mined by agarose
gel electrophoresis, indicating that there are three PstI sites in the linear fragment. The size of the pieces indicates the distance between the restriction sites. A second cut with BamHI
(bottom) yields two fragments, indicating one site. Since one BamHI
fragment (E) is very small, the BamHI site must be near one end of the fragment. Cutting with both enzymes indicates that the BamHI site is in the PstI fragment A.
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Restriction Enzyme Mapping
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Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
 Under nonstandard conditions, some restriction enzymes will bind to and cut sequences other than their defined recognition sequence. This altered specificity is called star activity. The propensity for star activity varies among enzymes. Thus, the nature and degree of star activity depends on the enzyme and the reaction conditions.
 Reaction conditions that induce star activity include suboptimal buffer, contamination with solvents or high concentrations of glycerol, prolonged reaction time, high concentration of enzymes, and divalent cation
imbalance.
 Restriction fragment length polymorphisms (RFLPs). RFLPs were the basis of the first molecular‐based human identification and mapping methods. Copyright © 2012 F.A. Davis Company
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Hybridization Technologies

Procedures performed in the clinical molecular laboratory are aimed at specific targets in genomic DNA. This requires visualization or detection of a particular gene or region of DNA in the backdrop of all other genes. There are several ways to find a target region of DNA.




Hybridization Technologies
Hybridization Method Southern blot
Northern blot
Target
DNA
RNA
Probe
Nucleic acid Nucleic acid 
Western blot
Protein
Protein

Southwestern blot
Protein DNA

Eastern blot
Protein
Protein

Far‐eastern blot
Lipids
(None)
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Purpose
Gene structure
Transcript structure, processing, gene expression Protein processing, gene expression DNA binding proteins, gene regulation
Modification of Western blot using enzymatic detection
(PathHunterTM); also, detection of specific agriculturally important proteins
Transfer of HPLC‐separated lipids to PVDF membranes for analysis by mass spectometry
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Southern Blot
The Southern blot
procedure allows
analysis of any
specific gene or
region without
having to separate it
from a complex
background.
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Restriction enzyme
digest
Genomic DNA
Gel electrophoresis
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
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Southern Blots
 Restriction Enzyme Cutting and Resolution



appropriate restriction enzyme
complete cutting of all sites
electrophoresis
 Preparation of Resolved DNA for Blotting (Transfer)
 Depurination
 larger fragments (>500 bp) are more efficiently denatured if they are depurinated before denaturation.
 the gel is first soaked in dilute hydrogen chloride (HCl) solution, a process that removes purine bases from the sugar‐phosphate backbone. This will “loosen up” the larger fragments for more complete denaturation.
 Denaturation
 DNA is denatured by exposing it in the gel to the strong base sodium hydroxide (NaOH)
 Blotting (Transfer)

10X SSC: 1 .5 M NaCl, 0.1 5 M Na citrate
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Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
 An apurinic site in double‐stranded DNA. Loss of the guanine (right) leaves an open site but does not break the sugarphosphate
backbone of the DNA
 A/G
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Southern Blot: DNA Binding Media
 Electrostatic and hydrophobic
 Nitrocellulose
 Nylon
 Reinforced nitrocellulose
 Electrostatic
 Nylon, Nytran
 Positively charged nylon
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Southern Blot: Capillary Transfer
Nitrocellulose
membrane
Soaked
paper
Dry
paper
Gel
Reservoir
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Southern Blot: Electrophoretic Transfer
Whatman
paper
-
Buffer
Buffer
Gel
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Nitrocellulose filter
Glass plates
+
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Southern Blot: Vacuum Transfer
Gel
Nitrocellulose filter
Recirculating
buffer
Vacuum
Porous plate
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Immobilization and Prehybridization  After transfer, the cut, denatured DNA is avidly bound to the membrane. The DNA can be permanently immobilized to the membrane by baking in a vacuum oven (80°C, 30–60 minutes) or by uv cross‐linking, that is, covalently attaching the DNA to the nitrocellulose using UV light energy
 Following immobilization of the DNA, a prehybridization step is required to prevent the probe from binding to nonspecific sites on the membrane surface, which will cause high background noise.  Prehybridization involves incubating the membrane in the same buffer in which the probe will subsequently be introduced or in a specially formulated prehybridization buffer. At this point, the buffer does not contain probe.  Blocking agents as Denhardt solution (Ficoll, polyvinyl pyrrolidane, bovine serum albumin) and salmon sperm DNA. Sodium dodecyl sulfate (SDS, 0.01 %) may also be included, along with formamide, the latter especially for RNA probes.
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Southern Blot: Probe
 DNA or RNA
 Covalently attached signal molecule
 Radioactive (32P, 33P, 14C)
 Nonradioactive (digoxigenin, biotin, fluorescent)
 Specific (complementary) to target gene
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Southern Blot Probe: Complementary Sequences
 The probe must be complementary to the target region of DNA.
 Complementary sequences are not identical.
 Complementary strands are antiparallel.
OP5′ ‐ GTAGCTCGCTGAT ‐ 3′OH
OH3′ ‐ CATCGAGCGACTA ‐ 5′OP
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Southern Blot: Probe
The probe determines what fragments are seen on the blot. Copyright © 2012 F.A. Davis Company
Molecular Diagnostics Fundamentals, Methods and Clinical Applications
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Melting Temperature (Tm)
 The temperature at which 50% of a nucleic acid is hybridized to its complementary strand.
DS
DS = SS
SS
Tm
Increasing temperature
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Tm in solution is a function of




Length of DNA GC content (%GC)
Salt concentration (M)
Formamide concentration
Tm = 81.5°C + 16.6 logM + 0.41 (%G + C) – 0.61 (%formamide) – 600/n
(DNA:DNA)
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Tm
For short (14–20 bp) oligomers:
Tm = 4° (GC) + 2° (AT)
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Stringency
 Stringency describes the conditions under which hybridization takes place.
 Formamide concentration increases stringency.
 Low salt increases stringency.
 Heat increases stringency.
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Stringency
Filter with
bound DNA
Probe in solution
Stringency too low
Stringency too high
Sequence-specific hybridization
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Southern Blot: Detection of Bound Probe

Radioactive or chemiluminescent detection requires exposure of the blot to autoradiography film.

Chromogenic detection occurs directly on the blot.

In end labeling, labeled nucleotides are added to the end of the probe using terminal transferase or T4 polynucleotide kinase. 
In nick translation, the labeled nucleotides are incorporated into single‐stranded breaks, or nicks, that are substrates for nucleotide addition by DNA polymerase. The polymerase uses the intact complementary strand for a template and displaces the previously hybridized strand as it extends the nick. 
Random priming generates new single‐stranded versions of the probe with the incorporation of the labeled nucleotides. The synthesis of these new strands is primed by oligomers of random sequences that are 6 to 10 bases in length. These short sequences will, at some frequency, complement sequences in the denatured probe and prime synthesis of copies of the probe sequences with incorporated Copyright labeled nucleotides.
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Molecular Diagnostics Fundamentals, Methods and Clinical Applications
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Radioactive Signal Detection
Filter with bound DNA
Radioactive isotope
Probe
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Nonradioactive Signal Detection
Substrate
Color or light
Antidigoxigenin antibody or avidin conjugated
to alkaline phosphatase or horseradish peroxidase
Probe covalently attached to digoxigenin or biotin
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Southern Blot Results
Radioactive or
chemiluminescent detection
(autoradiography film)
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Chromogenic detection
(nitrocellulose membrane)
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Southern Blot Applications
 Genetics, oncology (translocations, gene rearrangements)
 Typing/classification of organisms
 Cloning/verification of cloned DNA  Forensic, parentage testing (RFLP, VNTR)
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Northern Blot
 RNA structure and quantity
 Similar rationale as Southern blot except
 RNA target rather than DNA
 No restriction digestion required
 Probe must be designed as complement to the single‐
stranded RNA  Provides measure of relative expression of genes normalized to internal control
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 (up to approximately 30 µg total RNA or 0.5–3.0 g polyA
RNA
 Gel electrophoresis of RNA must be carried out under denaturing conditions
 Denaturant such as formaldehyde must be removed from the gel before transfer because it inhibits binding of the RNA to nitrocellulose. This is accomplished by rinsing the gel in deionized water
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Western Blot
 Serum, cell lysate, or protein extract is separated on SDS‐polyacrylamide gels (SDS‐PAGE) or isoelectric focusing gels (IEF). The former resolves proteins according to molecular weight, and the latter according to charge.
 Samples are treated with denaturant, such as mixing 1:1 with 0.04 M Tris HCl, pH 6.8, 0.1% SDS. 1 –50 µg of protein is loaded per well
 5%–20% polyacrylamide gels Copyright © 2012 F.A. Davis Company
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Western Blot  Proteins may be renatured before blotting to optimize antibody (probe)‐epitope binding.
 Proteins are blotted to membranes by capillary or electrophoretic transfer.  Probes are specific binding proteins, polyclonal antibodies, or monoclonal antibodies.
 Nitrocellulose has high affinity for proteins and is easily treated with detergent (0.1% Tween 20 in 0.05 M Tris and 0.15 M sodium chloride, pH 7.6) to prevent binding of the primary antibody probe to the membrane itself (blocking) before hybridization.
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Western Blot Signal Detection
Label
Primary
antibody
(probe)
Target
protein
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Secondary
antibody
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 The western blot method is used to confirm enzyme‐linked immunoassay results for human immunodeficiency virus (HIV) and hepatitis C virus among other organisms.
 known HIV proteins are separated by electrophoresis and transferred and bound to a nitrocellulose membrane.  The patient’s serum is overlaid on the membrane, and antibodies with specificity to HIV proteins bind to their corresponding protein antigens.  Unbound patient antibodies are washed off, and binding of antibodies is detected by adding a labeled antihuman immunoglobulin antibody.  If HIV antibodies are present in the patient’s serum, they can be detected with antihuman antibody probes appearing as a dark band on the blot corresponding to the specific HIV protein to which the antibody is specific.
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 Polyclonal antibodies are products of a generalized response to a specific antigen, usually a peptide or protein.
 Polyclonal antibodies are comprised of a mixture of immunoglobulins
directed at more than one epitope (molecular structure) on the antigen
 Monoclonal antibodies are more difficult to produce
 In western blot technology, polyclonal antibodies can give a more robust signal, especially if the target epitopes are partially lost during electrophoresis and transfer. Monoclonal antibodies are more specific and may give less background noise; however, if the targeted epitope is lost, these antibodies do not bind and no signal is generated.
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Filter‐Based Hybridization Technologies
Target
Probe
Southern blot
DNA
nucleic acid
Northern blot
RNA
nucleic acid
Western blot
protein
protein
Southwestern blot
protein
DNA
Eastern blot
protein
lectin, protein
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Other Blotting Formats ‐Array‐Based Hybridization
 Dot/Slot blots
 Dot blots
 Amplification analysis, Expression analysis (RNA), Mutation analysis
 Reverse dot blots
 Slot blots
 Amplification analysis, Expression analysis
 Genomic Array Technology
 Array technology is a type of hybridization analysis allowing simultaneous study of large numbers of targets (or samples). Arrays are applied to gene (DNA) amplification or deletion on comparative genome hybridization arrays and to gene expression (RNA or protein) analysis on expression arrays.
 macroarrays, microarrays, high‐density oligonucleotide arrays
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 Macroarrays are reverse dot blots of up to several thousand targets on nitrocellulose membranes.
 Microarray ‐ Tens of thousands of targets can be screened simultaneously in a very small area by miniaturizing the deposition of droplets
 High‐density oligonucleotide arrays and are used for mutation analysis, single nucleotide polymorphism analysis, and sequencing.
 Sample preparation for array analysis requires fluorescent labeling of the test sample, as microarrays and other high‐density arrays are read by automated fluorescent detection systems. The most frequent labeling method used for RNA is synthesis of cDNA or RNA copies with incorporation of labeled nucleotides. For DNA, random priming or nick translation is used.
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Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
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Molecular Diagnostics Fundamentals, Methods and Clinical Applications
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 Expression arrays measure transcript or protein production relative to a reference control isolated from untreated or normal specimens.
 Comparative genome hybridization (array CGH) is designed to test DNA. This method is used to screen the genome or specific genomic loci for deletions and amplifications.
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Comparative Genomic Hybridization (CGH)
 Immobilized, denatured normal chromosomes
 Test and reference DNA are labeled by incorporation of nucleotides covalently attached to fluorescent dyes.
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(Test)
(Reference)
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Comparative Genomic Hybridization (CGH)
 The labeled DNA is hybridized to the normal chromosomes on a microscope slide.
(Amplification at this locus)
Normal reference DNA
Test sample DNA
(Deletion at this locus)
 Differences between normal and reference will be revealed.
 Amplification: test color dominates.
 Deletion: reference color dominates.
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Comparative Genomic Hybridization (CGH)
 Green signal (test more than reference) = amplification
 Red signal (reference more than test) = deletion
Test:Reference
0.5 1.0 1.5 2.0
Amplification
Deletion
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Bead Array Technology
 The probes may also be immobilized on beads, allowing hybridization of the targets in the bead suspension
 In order to distinguish specific probes carried on different beads, the beads are color‐coded with a fluorescent dye. The sample is then labeled with a different dye so that the combination of the target and bead fluorescent signals indicates the presence or absence of a specific target. This technology can be used for protein as well as nucleic acid targets. Clinical tests using Luminex systems are available for infectious diseases and tissue typing.
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Solution hybridization
 In solution hybridization, neither the probe nor the target is immobilized. Probes and targets bind in solution, followed by resolution of the bound products.  With the increasing interest in short interfering RNAs (siRNAs) and microRNAs (miRNAs), which are conveniently analyzed by this type of hybridization analysis, solution methods may come into more frequent use.
 Solution hybridization has been used to measure mRNA expression, especially when there are low amounts of target RNA. One version of the method is called RNase protection, or S1 mapping, for the S1 singlestrand–specific nuclease.
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Molecular Diagnostics Fundamentals, Methods and Clinical Applications
Second Edition
S1 mapping
 In S1 mapping, the labeled probe is hybridized to the target sample in solution. After digestion of excess probe by a single‐strand–specific nuclease, the resulting labeled, double‐
stranded fragments are resolved by polyacrylamide gel electrophoresis
 S1 mapping is useful for determining the start point or termination point of transcripts
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Gel mobility shift assay

Solution hybridization can also be applied to the analysis of protein‐protein interactions and to nucleic acid–binding proteins using a gel mobility shift assay

After mixing the labeled DNA or protein with the test material, such as a cell lysate, a change in mobility, usually a shift to slower migration, indicates binding of a component in the test material to the probe protein or nucleic acid
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Summary
 Restriction enzymes cut DNA at specific recognition sequences.
 DNA can be characterized by restriction enzyme mapping.
 Specific DNA regions in a complex mixture are characterized using Southern blot.
 Specific proteins in a complex mixture are characterized using western blot.
 Regions of genomic amplification or deletion are characterized using comparative genomic hybridization.
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